We have psp cas 9 2a puro and esp cas 9 1.1 vectors. We are performing T7 assays for mutation detection. Do we have the possibility to use positive control? Do we have the chance to estimate how many base pairs will be the extra band ? We also did the single cell isolation after transfection and then expand them. Is there a protocol we can apply as alternative to this stage?
Hello Ibrahim,
you can use the T7 or surveyor assays on your bulk transfected cells. Instead of one PCR-band you should see two shorter ones corresponding to the length fractions caused by Cas9 DSBs in your amplicon.
Using your single cells you can do sanger sequencing with your PCR amplicon. If your gene has a protein product and a good AB is available you could/should check knock-out by western blot.
Hope that helps.
Hi Ibrahim,
I strongly suggest the Surveyor assay to detect your mutations that are present within your DNA. To design your primers for amplification of the region where you suspect your mutations, I suggest the Cosmid crispr software (https://crispr.bme.gatech.edu/). I've tried it for several genes and it works just fine. There are several brands for surveyor assay so just pick the one that is best suited for you. You can also directly sequence your amplicon for further sequencing which will you give information on the indel mutation.
Hello Ibrahim,
which cells are you working with? I would strongly suggest to test your gRNAs in HEK cells, as these cells are easy to transfect (tell me, if you need a protocol for this). If you have additional PCR-bands, you can go on with single cell dilution. In my experience, it doesn´t make sense to sequence the pool-DNA, as this could result in false-negative results (if the gene editing efficiency is too low).
As an alternative, you could use the vector PX458, which expresses GFP. Using this, you would be able to FACS-sort your cells.
After transfection, is there any other way for mutation imaging technique other than single cell isolation? we are that we have a mix population. Is it helpful to isolate genomic DNA after transfection and look at the t7 endonuclease assay?
- Badges
- Science method